Cancer-wide in silico analyses using differentially expressed genes demonstrate the functions and clinical relevance of JAG, DLL, and NOTCH

Notch ligands [jagged (JAG) and, delta-like (DLL) families] and receptors [NOTCH family] are key regulators of Notch signaling. NOTCH signaling contributes to vascular development, tissue homeostasis, angiogenesis, and cancer progression. To elucidate the universal functions of the JAG, DLL, and NOTCH families and their connections with various biological functions, we examined 15 types of cancer using The Cancer Genome Atlas clinical database. We selected the differentially expressed genes (DEGs), which were positively correlated to the JAG, DLL, and NOTCH families in each cancer. We selected positive and negative hallmark signatures across cancer types. These indicated biological features associated with angiogenesis, hypoxia, KRAS signaling, cell cycle, and MYC targets by gene ontology and gene set enrichment analyses using DEGs. Furthermore, we analyzed single-cell RNA sequencing data to examine the expression of JAG, DLL, and NOTCH families and enrichment of hallmark signatures. Positive signatures identified using DEGs, such as KRAS signaling and hypoxia, were enriched in clusters with high expression of JAG, DLL, and NOTCH families. We subsequently validated the correlation between the JAG, DLL, and NOTCH families and clinical stages, including treatment response, metastasis, and recurrence. In addition, we performed survival analysis to identify hallmark signatures that critically affect patient survival when combining the expression of JAG, DLL, and NOTCH families. By combining the DEG enrichment and hallmark signature enrichment in survival analysis, we suggested unexplored regulatory functions and synergistic effects causing synthetic lethality. Taken together, our observations demonstrate the functions of JAG, DLL, and NOTCH families in cancer malignancy and provide insights into their molecular regulatory mechanisms.


Compare JAG, DLL, NOTCH expression between primary tumor and metastasis
Step 4-3

GO analysis and GSEA with the DEGs
Step 6 Divide the patients into high/low group by JAG, DLL, NOTCH expression or DEG enrichment scores Step 6-1

Patient survival analysis by JAG, DLL, NOTCH expression
Step 6-4 Patient survival analysis -Signature enrichment -Signature enrichment combined with the expression or DEG enrichment score Step 6-2

Patient survival analysis by DEG enrichment scores
Step 6-3 Select the signatures with the highest significance S1 Fig. Information of the clinical datasets and overall scheme of our study.
(A) A table showing the information of the datasets, such as sources, gene counts, and the number of patients included in the dataset.(A) Box plots comparing the gene expressions of the JAG, DLL, and NOTCH families in BIC (GSE65194) and OV (GSE69428) and their corresponding normal tissues.Data were analyzed using a two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).(B) Box plots comparing the protein expressions of the JAG, DLL, and NOTCH families in BIC, CRC, GBM, HNSC, KRCC, LHC, LGG, LUAD, OV, and UCEC and their corresponding normal tissues.Data were analyzed using a two-tailed Student's t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

( B )
Workflow of the overall bioinformatic analysis.
of mRNA expression of JAG, DLL, and NOTCH families according to tumor stage.Box plots showing the gene expression of (A) JAG1, (B) JAG2, (C) DLL1, (D) DLL3, (E) DLL4, (F) NOTCH1, (G) NOTCH2, (H) NOTCH3, and (I) NOTCH4 according to tumor stage. of enrichment score of DEG for each JAG, DLL, and NOTCH families according to tumor stage.Box plots showing the enrichment score of (A) JAG1 DEG, (B) JAG2 DEG, (C) DLL1 DEG, (D) DLL3 DEG, (E) DLL4 DEG, (F) NOTCH1 DEG, (G) NOTCH2 DEG, (H) NOTCH3 DEG, and (I) NOTCH4 DEG according to tumor stage.showing the FDR q-value of GO analysis results.Each GO term category is included in biological processes and cellular components.
binding Positive regulation of angiogenesis Regulation of blood pressure RNA binding Positive regulation of cell migration Receptor complex Sarcolemma RNA polymerase II regulatory region sequence-specific DNA binding Protein serine/threonine kinase activity Positive regulation of vasculogenesis RNA polymerase II core promoter proximal region sequence-specific DNA binding Vascular endothelial growth factor signaling pathway Negative regulation of endothelial cell proliferation Negative regulation of angiogenesis Response to hypoxia Muscle organ development Transcription coactivator activity Microtubule Positive regulation of GTPase activity Platelet activation Positive regulation of phospholipase C activity Notch signaling pathway Positive regulation of MAP kinase activity Regulation of cell adhesion Rho guanyl-nucleotide exchange factor activity Vascular endothelial growth factor binding Beta-catenin binding Phosphatase regulator activity Sprouting angiogenesis Small cell lung cancer Rab guanyl-nucleotide exchange factor activity Transforming growth factor beta receptor signaling pathway Positive regulation of cytosolic calcium ion concentration Plasma membrane Positive regulation of endothelial cell proliferation Protein kinase binding Regulation of cell shape Positive regulation of cytosolic calcium ion concentration Transforming growth factor beta binding Protein complex Actin binding Transcription corepressor activity Positive regulation of endothelial cell migration Positive regulation of chemotaxis Transmembrane receptor protein tyrosine kinase activity Regulation of small GTPase mediated signal transduction Patterning of showing the FDR q-value of GO analysis results.Each GO term category is included in molecular functions and KEGG pathway.
DEG-based GO analysis using scRNA-seq in GBM.(A) Heatmap showing the expression of JAG, DLL, and NOTCH families in each cluster.(B and C) Bubble plot demonstrating GO term enrichment in (B) Cluster 10 and (C) Cluster 0. The bubble size represents the number of DEGs in each GO term.GO terms [-log (FDR value) < 2.5] are listed in the table below.
tSNE plot of LHC in clusters.(B) Log-normalized expression of JAG, DLL, and NOTCH families.(C) Heatmap showing the expression of JAG, DLL, and NOTCH families in each cluster.(D) Heatmap showing the enrichment of hallmark signatures in each cluster.D) tSNE plot of (A) BIC and (D) GBM in clusters.(B, E) Log-normalized expression of JAG, DLL, and NOTCH families in (B) BIC and (E) GBM cells.(C, F) Heatmap showing the expression of JAG, DLL, and NOTCH families in each cluster of (C) BIC and (F) GBM.S11 Fig. Box plots showing the expression of (A) JAG2, (B) NOTCH1, (C) NOTCH2, (D) NOTCH3, and (E) NOTCH4 in primary and metastatic tumors.Data were analyzed using a two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).plots comparing the expression of JAG, DLL, and NOTCH families in patients with anti-cancer drugs.(A) Expression of JAG, DLL, and NOTCH families before and after chemotherapy in BIC.Data were analyzed using a two-tailed Student's t-test (*p < 0.05, ***p < 0.001).(B) Expression of JAG, DLL, and NOTCH families before and after chemotherapy in CRC.(C) Expression of JAG, DLL, and NOTCH families before, 3 months and 6 months after chemotherapy with pazopanib in KRCC.Data were analyzed using a two-tailed Student's t-test.(D) Expression of JAG, DLL, and NOTCH families before and after chemotherapy with dabrafenib or vemurafenib in SCC. mRNA expression of Notch ligands between primary and recurrent tumors of each patient with BIC, GBM, LGG, OV, SCC, and TC.Scatterplots for Notch ligands including (A) JAG1, (B) JAG2, (C) DLL1, (D) DLL3, and (E) DLL4.mRNA expression of Notch receptors between primary and recurrent tumors of each patient with BIC, GBM, LGG, OV, SCC, and TC.Scatterplots for Notch receptors including (A) NOTCH1, (B) NOTCH2, (C) NOTCH3, and (D) NOTCH4.S15 Fig. Box plots showing the enrichment score of (A) JAG1 DEG, (B) JAG2 DEG, (C) DLL1 DEG, (D) DLL3 DEG, and (E) DLL4 DEG in primary and metastatic tumors.Data were analyzed using a two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).DEG, and (D) NOTCH4 DEG in primary and metastatic tumors.Data were analyzed using a two-tailed Student's t-test (*p < 0.05, ***p < 0.001).plots comparing the enrichment score of DEG for each JAG, DLL, and NOTCH families in patients with anti-cancer drugs.(A) Enrichment score of DEG for each JAG, DLL, and NOTCH families before and after chemotherapy in BIC.Data were analyzed using a two-tailed Student's t-test (*p < 0.05, ***p < 0.001).(B) Enrichment score of DEG for each JAG, DLL, and NOTCH families before and after chemotherapy in CRC.Enrichment score of DEG for each of JAG, DLL, and NOTCH families before and after radiotherapy and chemotherapy with cetuximab and AMG479 in HNSC.(D) Enrichment score of DEG for each JAG, DLL, and NOTCH families before, 3 months, and 6 months after chemotherapy with pazopanib in KRCC.(E) Enrichment score of DEG for each JAG, DLL, and NOTCH families before and after chemotherapy with IFNa-2b in LHC.(F) Enrichment score of DEG for each JAG, DLL, and NOTCH families before and after chemotherapy with dabrafenib or vemurafenib in SCC.Data were analyzed using a two-tailed Student's t-test.(*p< 0.05).S18 Fig. Tracking enrichment score of DEG for each Notch ligand between primary and recurrent tumors of each patient with BIC, GBM, HNSC, LGG, LHC, OV, SCC, and TC.Scatterplots including (A) JAG1 DEG, (B) JAG2 DEG, (C) DLL1 DEG, (D) DLL3 DEG, and (E) DLL4 DEG.
enrichment score of DEG for each Notch receptor between primary and recurrent tumors of each patient with BIC, GBM, HNSC, LGG, LHC, OV, SCC, and TC.Scatterplots including (A) NOTCH1 DEG, (B) NOTCH2 DEG, (C) NOTCH3 DEG, and (matrix showing the overall result from the survival analysis using the positive and negative signatures.The difference in survival, of which the p value from the log-rank t-test is less than 0.05, was presented to cause worse or better outcome.Survival analysis using gene expression or DEGs along with the gene signatures correlated to JAG-NOTCH signaling pathway.

Gene network analysis depicts the interactions between JAG, DLL, NOTCH genes and the DEGs, in the regulatory network of the associated signatures.
Gene network plots for each signature including (A) Notch signaling, (B) Angiogenesis, (C) Hypoxia, and (D) KRAS signaling UP.Black dots indicate the genes belonging to the DEGs, which are involved in the regulatory network of the signature.Grey dots denote the mediator which are not included in the DEGs but essential for the regulation of the signature.(E) A table showing the compositions of each interaction category described in the gene network analysis.